AND USE OF SUCH PROTEIN PARTICLES

Field of the invention

The present invention relates to a process for preparing protein particles, protein particles obtainable by such process, a dispersion of such protein particles, food products comprising such protein particles and use of such protein particles.

Background of the invention

There is a need for food products with a high protein content, in particular for liquid food products such as clinical food or high protein drinks such as sport drinks. It is, however, difficult to produce food products with a high protein content that are heat and/or shelf stable. Since proteins typically show denaturation behaviour upon heat treatment, liquids with a relatively high protein content will typically form a gel upon heat treatment. Also, many high protein liquids show undesired shear thickening behaviour, in particular after heat treatment.

In the prior art, native micellar casein is typically used as protein to prepare heat stable liquid food products. In WO2009/072885 is disclosed a liquid enteral nutritional composition comprising 6 to 14 grams of protein per 100 ml wherein the protein includes micellar casein and caseinate. Since micellar casein is quite bulky, it is difficult to manufacture food products with micellar casein with a high protein content. A further disadvantage of micellar casein is that it is not stable at low pH, since gelation of micellar casein already occurs at a relatively high pH value.

The use of denatured, heat stable protein particles in liquid food products has been described. In WO2010/077137 for example, a process for the preparation of denatured and gelled protein particles is described. Dispersions of protein particles prepared according to the process as described in WO2010/077137 appear, however, not to be heat stable in the sense that they show shear thickening behavior after being subjected to a heat treatment.

In patent application US2005/0255213 the preparation of artificial caviar has been described. To prepare such caviar droplets of a proteinaceous composition are fed to a heated oil bath. The proteins present in the drops of the proteinaceous composition gelatinize and grains are formed with a diameter of 1-3 mm. The oil does not comprise significant amounts of emulsifier, hence an emulsion of the proteinaceous composition in the oil is not formed.

Patent application US6767575 relates to a process for preparing denaturated whey protein aggregates. The process comprises the provision of an aqueous solution of at most 4 wt.% whey proteins, which solution is subsequently heated to 75-150° which causes denaturation of the whey proteins. The use of a liquid in-oil emulsion has not been described. Furthermore, protein content of the particles obtained is relatively low as well as their heat-stability.

Summary of the invention

A novel process has been found by which protein particles can be obtained with a high protein content, a good heat stability, and advantageous rheological properties. It has been found that dispersions of the protein particles obtained by the novel process do not show shear thickening behaviour after heat treatment.

Accordingly, the invention relates to a process for preparing protein particles, the process comprising the following steps:

(a) providing a dispersion of a protein in a liquid;

(b) providing an oil comprising an emulsifier;

(c) preparing a liquid-in-oil emulsion comprising dispersed droplets of the dispersion of protein in the oil by adding the dispersion of protein provided in step (a) to the oil provided in step (b) and mixing the dispersion with the oil;

(d) treating the emulsion obtained in step (c) to cause aggregation of the protein to obtain solidified protein particles in an oil phase; and

(e) separating the solidified protein particles from the oil phase.

In a further aspect, the invention relates to protein particles obtainable by a process as hereinabove defined.

Important advantages of the protein particles according to the invention are that they have a high protein content, are heat stable and have functional properties, in particular rheological properties that are to a large extent independent of the type and amount of protein. In particular, the protein particles do not show, if applied as a dispersion, shear thickening behaviour, even not after heat treatment. Accordingly, the invention further relates to a dispersion of protein particles as hereinabove defined in an aqueous phase.

A further advantage of the dispersion according to the invention is that such dispersion is stable, in particular heat and shelf stable, irrespective of the source of protein used.

Because of their favourable properties, such as high protein content and heat stability, the protein particles can advantageously be used in food products, for example to increase the protein content in a food product without jeopardizing the structure and functionality of the food product. Alternatively, the protein particles can suitably be used to manufacture food products with a relatively low protein content. The protein particles can advantageously be used to control the bulk properties of an end product such as for example a food product. In particular, the rheological profile and the protein content of a food product can be controlled by tuning the amount of protein particles used.

Accordingly, the invention relates in an even further aspect to a food product comprising the protein particles as hereinbefore defined. The food product may comprise the protein particles as such or in the form of a dispersion of the protein particles in an aqueous phase. The food product according to the invention may be such dispersion as such. Alternatively, the food product may comprise such dispersion and additional further food ingredients.

The protein particles according to the invention can suitably be used for increasing the protein content of a food product. Accordingly, the invention relates in a still further aspect to use of the protein particles as hereinabove defined for increasing the protein content of a food product.

The protein particles or the dispersion according to the invention may also be used in non-food products such as for example paints or coatings.

Summary of the drawings

In Figure 1 is shown SEM images of protein particles according to the invention (lower picture) and protein particles not according to the invention (upper picture).

In Figure 2 is shown the viscosity as a function of shear rate before and after heat treatment for a dispersion with protein particles according to the invention (2b) and for a dispersion with protein particles not according to the invention (2a). In Figure 3 is shown SEM images of macroscopic gels and of protein particles (microscopic gels) prepared from a dispersion of protein (according to the invention; lower images) and prepared from a solution of protein (not a according to the invention; upper images).

Detailed description of the invention

In the process according to the invention, first a dispersion of a protein in a liquid is provided (step (a)). The liquid may be any liquid that is immiscible with oil so that a liquid-in-oil emulsion can be formed in step (c). Preferably, the liquid is an aqueous liquid, more preferably the liquid is water. In the liquid, one of more proteins are dispersed. Any protein that can be caused to aggregate in step (d) of the process according to the invention may suitably be used. Preferably, the protein is a protein in its un-denatured state, i.e. in its non-aggregated form and still capable of aggregating in step (d).

The dispersion of protein provided in step (a) is a dispersion of insoluble protein in a liquid. Such dispersion may for example be a dispersion of protein in a liquid at conditions of pH, salt concentration, liquid composition and/or temperature such that the protein is at least partly insoluble.

Preferably, the dispersion is a dispersion of protein in a liquid having a pH value close to the iso-electric point of the protein. The pH is so close the the iso-electric point of the protein that at least part of the protein is insoluble in the liquid. Preferably, the pH is in the range of from 1.0 below the iso-electric point of the protein to 1.0 above the iso-electric point of the protein, more preferably of from 0.7 below the iso-electric point of the protein to 0.7 above the iso-electric point of the protein, even more preferably of from 0.5 below the iso-electric point of the protein to 0.5 above the isoelectric point of the protein, still more preferably of from 0.2 below the iso-electric point of the protein to 0.2 above the iso-electric point of the protein. The iso-electric point of a protein is the pH at which the protein has on average no net charge. Since the protein has no net charge, it will not be dissolved at or close to its iso-electric point. The dispersion may for example be provided by first dissolving the protein in the liquid to obtain a protein solution and then adjusting the pH of the solution to a pH value close to the iso-electric point of the protein so that the protein will become at least partly insoluble and a dispersion of the protein in the liquid will be formed. The dispersion may comprise more than one proteins. In case the dispersion comprises more than one proteins, the iso-electric point is defined as the iso-electric point of the protein that is present in an amount above 50 wt% of the total amount of proteins in the dispersion. In case none of the proteins in the dispersion is present in an amount above 50 wt%, the iso-electric point is defined as the pH at which the total net charge of the proteins is lowest. It will be appreciated that in case the dispersion comprises more than one proteins, not all proteins need to be dispersed. One or more proteins may be dissolved in the liquid.

Alternatively, the dispersion may be provided by adding salt and/or a solvent to an aqueous solution of a protein to obtain a dispersion of insoluble protein.

Reference herein to proteins is to a polypeptide having at least 10 amino acid units. Preferably, the protein has at least 20 amino acid units, more preferably at least 50, even more preferably at least 100 amino acids.

The protein dispersion may suitably comprise any protein capable of forming an aggregate. Preferably, the protein is a protein that can form an aggregate by means of heat gelation, such as globular proteins, or a protein that can form an aggregate upon acidification, such as for example caseine and caseinate. Preferred globular proteins are whey protein, egg protein, hemoglobulin, globular vegetable proteins or a combination or two or more thereof. Whey protein is particularly preferred globular protein.

Another preferred source of proteins is skim milk powder, which powder comprises in general 20-40 wt.% of different proteins, such as casein and whey proteins. Hence, in accordance with the present invention, skim milk powder may be used for preparing a dispersion of protein in a liquid, preferably water, as mentioned in step a) of the present invention.

The amount of protein dispersed is such that the protein can be caused to aggregate in step (d) and will thus inter alia depend on the type of protein used. Whey proteins for example can be caused to aggregate as from a concentration of 8 to 9 wt%. Caseines and caseinates can be caused to aggregate at a much lower concentration, typically as from 2.5 wt%. For globular proteins, the protein dispersion preferably comprises at least 11 wt % dispersed protein, more preferably at least 20 wt%, based on the total weight of the dispersion.

For processability reasons, the dispersion preferably does not comprise more than 50 wt% dispersed protein, more preferably not more than 40 wt%. In step (b) of the process according to the invention, an oil comprising an emulsifier is provided. Reference herein to oil is to a liquid oily phase. The oil may comprise one or more oils, preferably food grade oils, that are liquid at a temperature in the range of from 20 to 80 °C, preferably of from 20 to 50 °C. Examples of suitable food-grade oils include vegetable oil such as corn oil, sunflower oil, soybean oil, canola oil, rapeseed oil, olive oil, walnut oil, or peanut oil, algal oil, fish oil, and molten animal fat. In addition, the oil may further comprise natural and/or synthetic lipid components, including but not limited to saturated and unsaturated fatty acids, glycerol, glycerides, phospholipids, glycolipids, phytosterol and/or sterol esters.

The oil comprises an emulsifier. The emulsifier may be any emulsifier that is to some extent soluble in the oil and is suitable of emulsifying the aqueous liquid into the oil. The emulsifier is preferably food-grade. Preferably, the emulsifier has a low hydrophilic-lipophilic balance (HLB) value. Examples of suitable emulsifiers are polyglycerol polyricinoleate (PGPR; E476), lecithin and esters of sorbitan that are known as "Spans". Polyglycerol polyricinoleate is a particularly preferred emulsifier. The emulsifier may be present in any functionally effective amount. For polyglycerol polyricinoleate, such functionally effective amount is typically in the range of from 1 to about 10 wt%, based on the weight of oil. Further, it is to be noted that commercially available oil preparations may comprise emulsifiers as contaminants. In such case, the contaminant may serve as the emulsifier.

In step (c), a liquid-in-oil emulsion is prepared by adding the dispersion of protein in the liquid provided in step (a) to the oil provided in step (b) and mixing the dispersion with the oil. Thus, an emulsion is obtained with the dispersion being the discontinuous phase and the oil being the continuous phase. The weight ratio of oil to liquid in the emulsion preferably is in the range of from 95% : 5% (w/w) to 45% : 55% (w/w). More preferably, in the range of from 10 to 55 wt% liquid is dispersed in the oil phase, based on the weight of the oil phase.

Thus, dispersed droplets of protein dispersion in an oil phase are formed.

Preferably, the mixing is performed such that the dispersed droplets of protein dispersion have a volume-to-surface mean particle diameter (d32 or Sauter mean diameter) in the range of from 0.1-20 μπι, more preferably of from 1 to 10 μπι. The volume-to-surface mean particle diameter may be determined by means known in the art such as direct light microscopy, confocal laser scanning microscopy, or static light scattering.

In step (d), the emulsion obtained in step (c) is treated to cause aggregation of the proteins. Aggregation of proteins typically occurs by cross-linking the proteins. Such cross-linking may for example be effected by heating, acidification, enzyme treatment, using chemical cross-linking agents or combinations thereof. Any suitable treatment that causes aggregation of the proteins may be applied. Preferably, in particular in case the proteins present in the protein dispersion are globular proteins, the emulsion is heated to a temperature above the thermal denaturation temperature of the protein. In case the protein is caused to aggregate by acidification, the emulsion is treated in step (d) to cause lowering of the pH of the dispersed droplets, for example by adding an acidifier, preferably an oil-soluble acidifier. Alternatively, a compound is used that slowly releases acid, such as for example glucono delta lactone. Such compound may for example be added to the starting dispersion. Step (d) then comprises allowing time to pass in order to allow the glucono delta lactone to release sufficient acid for aggregation to occur. Aggregation by means of enzymes may for example be carried out by adding enzymes to the starting dispersion of protein. The treating in step (d) may then comprise allowing time to pass in order to allow the enzymes to cause aggregation of the proteins.

Thus, particles of solidified protein are obtained. Typically, the protein in each droplet of protein dispersion will aggregate and each droplets thus forms a particle of solidified protein. Thus a dispersion of particles of solidified protein in an oil phase is formed. The size of the protein particles will typically be comparable to the size of the droplets in the emulsion, although shrinking or swelling effects may result in somewhat larger or smaller particles. Preferably, the protein particles thus obtained have an average diameter in the range of from 0.1 to 20 μπι, more preferably of from 1 to 10 μπι. It has been found that the particles thus-formed typically have an irregular shape.

The solidified protein particles thus-obtained are separated from the oil phase. Such separation may be done by any means known in the art, for example by first subjecting the treated emulsion to centrifugation to obtain a separate oil layer, followed by removal of the separate oil layer. Alternatively, oil may be removed by extracting the oil from the treated emulsion with a suitable extractant, for example hexane or supercritical carbon dioxide. Preferably, the oil is removed by first subjecting the treated emulsion to centrifugation and then removing the oil layer thus created. A pellet comprising protein particles is thus obtained. In order to remove remaining oil, the protein particles obtained after a first oil removal step (by centrifugation, extraction or otherwise) may be washed with water or another aqueous phase, optionally comprising an emulsifier, followed by a centrifugation step to separate and remove an oil layer or emulsified oil particles. Such washing and centrifugation steps may be repeated.

The protein content of the protein particles is preferably at least 5 wt%, more preferably 11 wt%, even more preferably at least 15 wt%, still more preferably at least 25 wt%, and still more preferably at least 35 wt%. The protein content is preferably at most 70 wt%, more preferably at most 60 wt%. It will be appreciated that the preferred protein content strongly depends on the concentration of dispersed protein in the dispersion provided in step (a) and on the type of protein used.

It has been found that protein particles obtained by the process according to the invention have a higher protein density than protein particles obtained by the method described in WO2010/077137, when starting with a solution (WO2010/077137) or dispersion (present invention) with the same content of proteins.

The invention further relates to a dispersion of protein particles according to the invention in an aqueous phase. Such dispersion may for example be obtained by dispersing the protein particles obtained after removal of oil from the treated emulsion in an aqueous phase. The aqueous phase may for example be water or an aqueous alcohol. Preferably, the aqueous phase is water.

The dispersing of protein particles may be done by any suitable techniques known in the art. Typically the aqueous phase will be added to the protein particles followed by subjecting the resulting mixture to sufficient agitation to obtain a dispersion, for example by high speed mixing of by passing the solution with protein particles through a homogenizer.

Preferably, the dispersion according to the invention has a viscosity in the range of from 1 to 100 mPa.s at a shear rate of 100 s ^"1 at 25 °C, more preferably of from 1 to 10 mPa.s at a shear rate of 100 s ^"1 at 25 °C.

The dispersion may comprise any suitable concentration of protein particles. It will be appreciated that the upper limit of the particle volume fraction will be determined by the content at which the dispersion will reach close packing, i.e the point at which the viscosity will diverge to infinity. Therefore, the dispersion may comprise up to 63 vol% protein particles, more preferably up to 40 vol%. Preferably, the dispersion comprises at least 10 vol%, more preferably at least 15 vol%, even more preferably at least 20 vol% protein particles. A content of in the range of from 15 to 40 vol% protein particles is particularly preferred.

The dispersion may comprise components other than protein particles and water. The dispersion may for example comprise further protein dissolved in the continuous aqueous phase, oil, or other food ingredients. The dispersion may have suitable protein content. Preferably, the total protein content of the dispersion is at least 7 wt%, more preferably at least 15 wt%, even more preferably at least 20 wt%.

The dispersion according to the invention may be a food product as such, for example a clinical food product such as a high protein nutritional composition for enteral administration. The dispersion may also form part of a food product that also comprises further ingredients.

Preferably the pH of the dispersion comprising the protein particles according to the present invention is more than 0.5 below or more than 0.5 above the iso-electric point of the protein. More preferably the pH of the dispersion is more than 1.0 below or more than 1.0 above the iso-electric point of the protein. At such pH very stable dispersions (e.g. heat stable) are obtained.

The food product according to the invention, i.e. a food product comprising the protein particles or the dispersion according to the invention, may comprise the protein particles or the dispersion of protein particles and additional further food ingredients, such as for example fat, carbohydrates, additional proteins and further nutrients such as for example vitamins, flavours and other additives.

The protein particles according to the invention may advantageously be used to increase the protein content of a food product, such as for example high-protein sport drinks. Alternatively, the protein particles may be used to manufacture food products with a very low protein content.

The food product preferably has a protein content (total of protein in the protein particles and further protein) of at least 7 wt%, more preferably at least 15 wt%, even more preferably at least 20 wt%.

An important advantage of the present invention is that a food product with a high protein content or a very low protein content can be obtained irrespective of the protein source that is used. Moreover, by using protein particles according to the invention, a heat stable dispersion is obtained irrespective of the protein source that is used.

The invention is further illustrated by means of the following non-limiting examples.

EXAMPLES

EXAMPLE 1 (Protein particles prepared from whey protein isolate) Preparation of protein particles

A solution of 25 wt% whey protein isolate (WPI) in water was prepared by dispersing a whey protein isolate powder (BiPro JE 034-7-440-1, ex. Davisco Foods International Inc., Minnesota, USA) comprising beta-lactoglobulin and alpha- lactalbumin in water and then stirring for 2 hours at room temperature. The protein solution was then stirred overnight at a temperature of 4 °C to achieve complete hydration. The pH of the solution was then adjusted to 5.5 using 6M HC1. Thus, a dispersion of whey protein in water was obtained.

An oil with an emulsifier was provided by dissolving 2.5 wt% polyglycerol polyricinoleate (PGPR 90, ex. Grindsted, Denmark) in food-grade sunflower oil by stirring at room temperature during 2 hours.

A water-in-oil emulsion was prepared by adding 10 grams of the 25 wt% WPI dispersion to 90 grams of the sunflower oil whilst mixing with a high speed mixer (Ultra-turrax T25, ex. IKA Werke, Germany) at a speed of 6,500 RPM and continuing mixing at this speed during 5 minutes. The water-in-oil emulsion was then heated to a temperature of 80 °C and maintained at this temperature for 20 minutes in order to cause gelation of the whey proteins. The emulsion was then centrifuged at a speed of 33,768 times g during one hour. After centrifugation, excess oil was removed by decanting the upper layer. The resultant pellet of protein particles was washed with a solution of 1 wt% whey protein isolate in water in order to disperse remaining oil in an aqueous continuous phase. The thus-obtained oil-in-water emulsion was centrifuged to separate oil droplets from the continuous phase and the upper layer of oil thus obtained was removed. The washing and oil removal was repeated two times (three washings in total).

Analysis of the protein particles obtained

The protein particles in the pellet obtained after the third centrifugation were analysed as follows:

The morphology was analysed by optical microscopy and by scanning electron microscopy (SEM). For SEM analysis, protein particles were diluted 1000 times in distilled water (milliQ water) and subsequently critical-point dried with carbon dioxide.

The protein content was calculated from the nitrogen content of the protein particles. Nitrogen content of the protein particles was measured by means of a Flash EA 112 N/protein analyzer (ex. Thermo Scientific, Waltham, USA).

Heat stability

Protein particles prepared in EXAMPLE 1 were dispersed in a solution of 1 wt% WPI in water. The content of protein particles in the dispersion was 31 vol%. The dispersion was subjected to a temperature of 90 °C during 30 minutes. The shear viscosity and the particle size distribution of the dispersions prior and after heat treatment were determined. The shear viscosity was determined by measuring the viscosity at different share rates by using a Physica MCR 501 Rheometer with a concentric cylinder (CC17/T200/SS; cup diameter of 18.88 mm; bob diameter of 16.66 mm). A sample of 5 ml of the dispersion was placed in the measuring cell of the rheometer and the surface of the sample was covered with paraffin oil. The shear viscosity of the samples was measured over the shear range of from 1 to 1,000 s ^"1 at 25 °C in duplicate. In Figure 2b is shown the viscosity (Pa.s) as a function of shear rate (s ^" ) of the dispersion before heat treatment (filled symbols) and after heat treatment (open symbols). The particle size distribution was determined by means of light scattering.

EXAMPLE 2 (comparison)

A solution of 25 wt% WPI in water was prepared as described in EXAMPLE 1, but now the pH value of the solution was left unadjusted at pH 6.8. Thus, a solution of protein was obtained. Protein particles were prepared from the WPI solution as described in

EXAMPLE 1 for the WPI dispersion. The protein particles thus obtained were analysed as described in EXAMPLE 1. The heat stability of a dispersion with 38 vol% protein particles in a 1 wt% WPI solution was determined as described in EXAMPLE 1. In Figure 2a is shown the viscosity (Pa.s) as a function of shear rate (s ^"1 ) at 25 °C for the dispersion before heat treatment (filled symbols) and after heat treatment (open symbols).

RESULTS OF EXAMPLES 1 AND 2

Morphology

Optical microscopy of the untreated emulsions, the heat-treated emulsions and the final protein particles showed that in both examples the untreated emulsion comprised droplets of a similar size (droplets with a diameter of a few microns). After heat treatment of the emulsions, irregular particles were formed in EXAMPLE 1 and spherical particles were formed in EXAMPLE 2. The size of the particles was similar to the size prior to heat treatment.

In Figure 1, SEM images are shown of the particles obtained in EXAMPLE 1

(lower picture) and EXAMPLE 2 (upper picture). Figure 1 shows that in EXAMPLE 2 (not according to the invention), spherical particle with a diameter of a few micrometer were formed. The particles obtained in EXAMPLE 1 (according to the invention) have a similar size, but an irregular, cauliflower-like shape.

Protein density

Determination of the protein content of the particles obtained in EXAMPLES 1 and 2 showed that the protein particles obtained in EXAMPLE 1 (according to the invention) had a protein content of 38.4 wt% whereas the protein particles obtained in EXAMPLE 2 (not according to the invention) had a protein content of 17 wt%.

Heat stability

The dispersion of particles obtained in EXAMPLE 2 (comparison) showed a slight increase in particle size after heat treatment. The dispersion with particles obtained in EXAMPLE 1 (particles according to the invention) did not show an increase in particle size.

In Figure 2, it can be seen that prior to heat treatment, both dispersions showed Newtonian behaviour. After heat treatment, the dispersion with the particles according to the invention still showed Newtonian behavior (no shear thickening), whereas the dispersion with the particles of EXAMPLE 2 had an increased viscosity and showed shear thickening at a shear rate of about 100 s ^"1 .

EXAMPLE 3

A macroscopic gel was prepared, both of the dispersion of WPI prepared in EXAMPLE 1 (pH 5.5) and of the solution of WPI prepared in EXAMPLE 2 (pH 6.8). The solution or dispersion was heated to a temperature of 80 °C and maintained at this temperature for 20 minutes in order to cause gelation of the whey proteins.

The morphology of the macroscopic gels thus formed was analysed by scanning electron microscopy (SEM).

In Figure 3 can be seen SEM images of the macroscopic gels thus formed and of the protein particles in the treated emulsions (microscopic gels) prepared according to EXAMPLES 1 and 2.

Upper left image : macroscopic gel of the pH 6.8 solution;

Upper right image: treated emulsion (microscopic gel) of the pH 6.8 solution;

Lower left image: macroscopic gel of the pH 5.5 dispersion; and

Lower right image: treated emulsion (microscopic gel) of the pH 5.5 dispersion.

The images show that the desired irregular structure of the protein particles according to the invention, i.e. prepared starting from a dispersion with a pH value close to the iso-electric point of the protein, can already be observed in a macroscopic gel of the starting dispersion. Therefore, preparing a macroscopic gel of the dispersion provided in step a) is a convenient and suitable means for testing whether a suitable protein dispersion has been used for preparing the protein particles according to the invention.

EXAMPLE 4 - (Protein particles prepared from Na-caseinate) Preparation of protein particles

10% (w/w) Na-caseinate dispersion was prepared by adding the Na-caseinate powder in Millipore water. The dispersion was stirred overnight (at 4°C). GDL was added at 1.5 or 3 wt%, resulting in a final pH of 4.6-4.7 and 3.6-3.7 respectively (after 24h). Directly after addition of GDL, the sample was stirred 2-3 min.

Subsequently, the dispersion was slowly added in Sun-flower oil (containing 2.5%) PGPR) while being mixed with a high speed mixer as described in Example-1. A total mixing time of 7 min (2 min addition of water phase into the oil phase + 5 min further mixing) at 6600 RPM was applied. The ratio of water to oil phase was kept at 2:8 during the first mixing step. After mixing, the emulsion was kept in dark for 24h, for complete acidification.

The following washing steps for the particles prepared at pH 3.6 were done in a 1%) (w/w) WPI solution as described in Example-1.

For the particles prepared at pH 4.6, dispersing in 1%> WPI (at pH 3.6) resulted in a yoghurt-like thick dispersion during homogenization step and no further oil removal during centrifugation was obtained. Samples were therefore mixed with a high speed mixer until a fine sample was obtained and centrifuged as described in Example-1. The final dispersing step for both samples was the same as in Example-1.

The final pH of the Na-caseinate particles prepared at pH 4.6-4.7 and dispersed in 1%) WPI (at pH 3.6) was 3.9. Before the heat stability analysis as described in

Example-2, the pH of this dispersion was acidified to 3.6 with 3M HCL.

Microscopy observation:

At both pH (i.e. pH 3.6-4.7 and pH 4.6-4.7) of preparation, protein particles in the size of a few microns were formed. Particles were spherical initially (w/o emulsion) and their final shape was irregular, suggesting that particles have shrunk and not swollen during preparation.

Internal protein content (Particles prepared at pH 3.6-3.7 and dispersed in 1% WPI at pH 3.6)

The internal protein content of the particles was calculated to be approximately 16-17% (w/w). Visual observation (particles prepared at pH 4.6-4.7 and dispersed in 1% WPI at pH 3.6)

The dispersion had a white, milk-like appearance both before and after heat treatment of the dispersion and no changes in the appearance (such as coagulation, viscosity increase or precipitation) was observed after heat treatment.

Heat stability (particles prepared at pH 4.6-4.7 and dispersed in 1% WPI at pH 3.6) The viscosity of the dispersion after heat treatment increased only to a very small extent (at a shear rate of lOs-1 it increased from 0.022 to 0.032 Pa.s). Furthermore, no swelling of the particles was observed. This suggests that the protein particles according to the present invention have a good heat stability.

Conclusion

Particles can be prepared from a 10% Na-caseinate dispersion by combination of cold gelation and two-step emulsification. At pH values either close to the isoelectric point of Na-caseinate (4.6) and lower than the isoelectric point, for example at pH 3.6, protein particles according to the invention could be prepared. The final shape of the protein particles was irregular and protein content was higher than the initial protein content of the Na-caseinate solution, suggesting shrinkage of the particles during preparation. Clearly, no swelling or a large viscosity increase was observed after heat treatment in the dispersions of particles prepared close to isoelectric point. Hence, the protein particles according to the present invention are considered heat stable.